There is a great need in the medical industry for blood substitutes and plasma volume expanders. Blood substitutes are useful for replacing blood lost by acute hemorrhage or during surgery, for supplying oxygen to tissues and organs and generally maintaining blood volumes. Plasma expanders are useful in volume deficiency shock, as an alleviant in anaphylactic and allergic shock and for replacing plasma lost following burn injuries. Donor blood banks have proved to be inadequate to meet this need for a number of reasons. Not only do blood banks often experience a shortage of donor blood, but blood that is donated can be unacceptable for medical use. Donor blood can be contaminated with hepatitis or the acquired immune deficiency syndrome (AIDS) virus, thereby posing significant risks to patients who receive the blood. In addition, donated blood has a relatively short shelf life.
In view of these problems, there has been extensive interest in the development of blood substitutes and blood plasma expanders which can be easily prepared and used in place of donated blood. To be effective, such a substitute should be characterized by a sufficiently high oxygen-binding capacity for use under normal environmental conditions. It also should not be subject to rapid renal elimination.
One type of substitute that has generated significant interest is modified hemoglobins. Natural mammalian hemoglobin is a tetramer, i.e., it is characterized by four polypeptide chains, two identical alpha chains and two identical beta chains, that are noncovalently linked together. In plasma, oxygenated hemoglobin has a tendency to split into dimers, each of which is small enough to be filtered by the kidneys and be excreted. Such dimers thus potentially cause renal damage and have significantly decreased intravascular retention time. As a result of the shortcomings of natural hemoglobin, efforts have been made to modify the hemoglobin molecule to make it resistant to dimerization.
Known treatments for crosslinking hemoglobin to make it resistant to dimerization involve reaction of hemoglobin with bifunctional reagents such as glutaraldehyde (C. Hopwood et al., Histochem J. 2, 137, 1970), glycolaldehyde (J. M. Manning, "Carboxymethylated Cross-Linked Hemoglobin A as a potential Blood Substitute", Abstract in the Program and Abstracts, Symposium on Oxygen Binding Heme Proteins Structure, Dynamics, Function and Genetics, Asilomar Conference Grounds, Pacific Grove, Calif., Oct. 9-13, 1988) or diimidate esters (W. Mock et al., Fed. Proc. 34, 1458, 1975, and U.S. Pat. No. 3,925,344). In U.S. Pat. No. 4,473,496, linear alpha-omega or heterocyclic polyaldehydes containing negatively charged groups are described as suitable for both decreasing the oxygen affinity of hemoglobin and for producing inter- and intramolecular crosslinking of hemoglobin. These reagents include carbohydrate-containing molecules such as mono- and polyphosphorylated nucleotides partially oxidized with periodate, so as to obtain aldehydic groups. The coupling reaction is based on the formation of Schiff bases of the aldehydic groups with the amino groups of the hemoglobin molecule. The schiff bases are then transformed into stable, covalent bonds by reduction with sodium or potassium borohydride, or another strong reducing agent. Finally, U.S. Pat. No. 4,584,130 describes the use of bifunctional crosslinking reagents such as diethyl 2,2'-sulfonyl-bis-malonate, ethyl 2,2'-sulfonyl-2,2'-benzenesulfonyl-bis-acetate, and the like, that have utility in crosslinking stroma-free hemoglobin to provide a modified hemoglobin having a physiologically acceptable oxygen affinity and suitable circulating half-life in vivo.
Another modified hemoglobin is described in U.S. Pat. No. 4,598,064 to Walder (1986). Specifically, Walder teaches crosslinking the two alpha chains of the tetrameric hemoglobin molecule, specifically at Lys 99 Alpha.sub.1 and Lys 99 Alpha.sub.2. A second patent by Walder, U.S. Pat. No. 4,600,531 (1986), discloses what the patentee describes as a process for the high level production of alpha, alpha-crosslinked hemoglobin. In accordance with this process, unmodified hemoglobin is deoxygenated and crosslinked, with the crosslinking occurring in the presence of an added polyanion which binds elecrostatically to deoxyhemoglobin at the 2,3-diphosphoglycerate binding site, located between the beta chains. This blocks side reactions of the crosslinker within this site and neighboring regions of the protein, thereby enhancing the chances of reaction at the desired Lys 99 Alpha.sub.1 and Lys 99 Alpha.sub.2 site, access to which is not blocked by the polyanion. Suitable polyanions are said to be 2,3-diphosphoglycerate, inositol hexaphosphate (IHP) and inositol hexasulfate, with IHP said to be preferred. The patent states that the concentration of polyanion should be within the range of from equimolar amounts with the hemoglobin to as much as a twenty molar excess, preferably from about 5 times to about 10 times the molar amount of hemoglobin.
In the examples section of the patent, Walder shows that, when bis(3,5-dibromosalicyl) fumarate was used as crosslinking agent in the absence of IHP, the yield of the desired crosslinked product was only about 5%. Approximately 10-15% of the hemoglobin was unmodified at the conclusion of the reaction period. Side reactions at other sites on the protein accounted for the remaining material. In the presence of 1.5 mM, however, as shown in example 3, the yield of alpha, alpha-crosslinked hemoglobin was found to be increased to 40%. At 5 mM IHP, the yield of alpha, alpha-crosslinked derivative was 60-65% with less than 6% impurities due to side reactions at other sites of the protein. Walder states that there were no further improvement in yield if the concentration of IHP was further increased.
Similarly, in example 5 of the Walder patent, under similar reaction conditions, at IHP concentrations of 1.3 and 1.5 mM, the yield was between 55% and 65%. At IHP concentrations of 2.0 mM or more, the yield of desired product was found to decrease progressively because of side reactions at other sites on the protein.
In addition to the limitations on product yield, the process described by Walder, although useful, has been found to have a number of other disadvantages. First, the use of IHP causes significant increases in the percentages of methemoglobins in the product mixture. These methemoglobins are not useful as blood substitutes of plasma expanders, nor do they have any other known clinical applications. The amount of methemoglobin formed can be decreased if the crosslinking reaction is carried out at temperatures of 25.degree. C. or lower. This has proved to be impractical, however, for it has been found that if the reaction is carried out at less than about 37.degree. C., the yield of the desired alpha, alpha-crosslinked protein is reduced, even when extended reaction times are used.
It also has been found that if the IHP is not removed from the product mixture after crosslinking is complete and prior to subsequent manipulations of the product mixture, oxidation of the various modified and unmodified hemoglobins to methemoglobins is accelerated relative to rates of methemoglobin formation in the absence of IHP. It is possible to remove the IHP from the reaction mixture once the crosslinking reaction has gone to completion but prior to subsequent manipulations of the product mixture. This procedure, however, adds a step, and, therefore, time and expense to the reaction process described by Walder. The additional step also increases the opportunity for chance microbial or pyrogen contamination of the product.
Yet another disadvantage of the Walder process is that, if the crosslinking reaction is completed using crude hemoglobin lysates, the yield of alpha, alpha-crosslinked hemoglobin is consistently less than the yield that is realized when purified hemoglobin lysates are used. When operating on a commercial scale, however, the use of crude hemoglobin lysates is preferred for hemoglobin modification processes.
Accordingly, improvements of the process disclosed by Walder are sought. It is an object of the present invention to provide a process for the formation of crosslinked hemoglobin in which the yields of desired product are enhanced in comparison to the yields obtainable using the prior art processes. It is a further object of this invention to develop such as process in which the levels of methemoglobins produced are relatively low.
Further objects of this invention will be apparent from reading the description of the invention set forth below and the appended claims.